This research grant concerns the ubiquitous calcium-binding protein calmodulin and its role in the regulation of cellular processes in the ciliated protozoa Paramecium tetraurelia. Calmodulin has been shown to play a key role in the control of the behavioral process in Paramecium; calmodulin mutants inhibit the activity of the Ca++-dependent K+ and Na+ channels. The current project intends to continue the use of those unique aspects of the Paramecium system to investigate the role of calmodulin in the regulation of calmodulin-dependent cellular phenotypes. There are three sections to the grant. 1) Site-specific mutagenesis of the wildtype calmodulin gene, in combination with transformation of the existing calmodulin mutants, will be used to study those aspects of the calmodulin molecule that are required for the regulation of a Ca++-dependent ion channel. The calmodulin protein from those mutations that are shown to adversely affect the regulation of the ion channels will be analyzed using several in vitro enzyme assays to determine the ability of the mutated calmodulin to control other processes. 2) 3'-modified antisense oligodeoxynucleotides complementary to calmodulin mRNA will be used as a means to study mutagenized calmodulin genes in a wildtype background. The antisense oligodeoxynucleotides reduce the level of the endogenous wildtype calmodulin, and this will allow the altered transforming calmodulin gene to be the dominant calmodulin in the cell. 3) The isolation and characterization of intragenic suppressors of the existing calmodulin mutants will be undertaken. The intragenic suppressors will provide information as to the requirements of the calmodulin structure. The combination of these three sections of the grant will address the structure-function properties of calmodulin in regards to specific cellular phenotypes using an in vivo system.